1. As mandated by federal regulations, investigators have an obligation to minimize pain, discomfort, and distress in animals used in their research. When mice are used in studies where wasting and death are potential complications, investigators and animals benefit from practical, rapid, and non- invasive methods for assessing health status and determining when euthanasia is indicated. Monitoring criteria used to make this determination are referred to as “endpoints” and are often developed to be specific for the manipulation performed with the model and the animal. Most endpoint criteria consist of a panel of clinical observations that suggest that an animal is in pain or distressed, requiring intervention through treatment or euthanasia. Weight is a commonly used assessment of animal well-being as it is a sensitive indicator of appetite and hydration status. Studies where tumor growth is induced are often very challenging as the growth of the tumor may mask loss of weight for the animal. A body condition scoring (BCS) technique has been characterized as a means of evaluating the health of mice. This study concluded that BCS has potential as an endpoint for mice on studies where weight is an unreliable indicator of mouse health, such as tumor growth and ascites studies, but that formal evaluation of this application should be pursued. Although many institutions report using BCS to assess mouse health clinically, follow-up studies have not been published. This study evaluated BCS as a monitoring criterion for skin and intra-abdominal tumor growth studies, used in conjunction with more traditional criteria such as weight loss, general body condition, attitude, tumor size, and the presence of ulceration on the tumor. Materials and Methods: Mice were palpated using previously published techniques. To assess body condition score, the mouse was placed on a flat surface (e.g. wire bar lid). Holding the base of the tail with the thumb and index finger of one hand, the degree of muscle and fat cover was assessed and assigned a score by palpating the sacroiliac region (Figure 1). At least two observers, blinded to the treatment of the mouse, were assigned to score the mice. All mice were scored on the same day, with each observer completing their scoring independently and within 6 hours of the other. The scoring involved weighing of the mouse, measurement of tumors (if visible), palpation to assign body condition score, and characterization of the mouse’s attitude (“curious” or “lethargic”) and posture (“normal” or “hunched”). These scores were entered into a spreadsheet for further analysis. A new score sheet was used each day to blind the observers to the values they had assigned to a particular mouse previously. Results: Initially, samples of all recorded parameters by each observer were compared to determine reproducibility of the body condition score technique. The differences between each observer’s weight and body condition score record were analyzed through examination of the mean and confidence intervals and z-tests (Microsoft Excel and JMP). For the mice on the abdominal tumor study, there was statistically significant variation between observer weights (p<0.05). For the mice on the skin tumor study, there was no significant between observer variation in weights (p=0.1013). Body condition scores for both studies had statistically significant between observer variation (p<0.05). However, the difference was found to be 0.08 ± 0.024 (mean ± CI). Discussion: The analysis of differences between observer assigned body condition scores and weight collection was of interest for demonstrating consistency of the techniques. The analysis was made with the assumption that there would be no difference between observers. The analysis revealed that there were differences between observers in weights and body condition score parameters, but no significant difference between measurements of tumors on animals in the skin tumor study. For the weights, the differences were suspected to be due to the interval of up to 6 hours between measurements, suggesting that weights must be consistently collected at the same time each day for appropriate interpretation of observations. The scale that was used for the abdominal tumor project was able to weigh out to thousandths of grams and was very sensitive to “noise” produced by animal movement. For the skin tumor project, a newer, less sensitive gram scale was used, resulting in the non-significant difference between each observer’s record of weights (p=0.9494), suggesting that equipment must also be considered when interpreting apparent weight loss or gain. Weight was not found to be a reliable predictor of animal health due to the number of potential variables that could affect accurate and consistent measurements. For the body condition scores, the average difference between each observer was 0.08 ± 0.024 (mean ± CI). Although the difference was statistically significant using a two-tailed z-test for analysis, the average difference is less than the 0.5 intervals of the body condition scoring system, suggesting that the difference is not of practical concern. This data supports the conclusion submitted by Ullman-Cullere (1999) that the technique is reproducible and consistent between individuals. We also found that if observers were asked to limit their score to integers without modifiers (“+” or “-”), the scores were more consistent between observers (data not shown). Methods: The mice for this study were the crossbred offspring of two transgenic lines of mice generated in the ICR strain. The first transgenic line was designated as gene- switch-TGFß1 mice. In this line, epidermal expression of TGF-ß1 can be specifically inhibited through the application of RU486. The second transgenic line was designated as ΔßRII-transgenic mice. In this line, the neonates exhibit a hyperproliferative epidermis that normalizes in adulthood. The crossbreeding of these two strains of mice resulted in TGF- ß1/ ΔßRII-transgenic mice where TGF- ß1 expression was inducible with constitutive ΔßRII expression. Briefly, crossbred TGF- ß1/ ΔßRII-transgenic mice and nontransgenic littermates were treated with a single topical application of dimethylbenzanthracene (DMBA) at week one. They also received weekly topical application of 12-0-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks, followed by RU486 (20μg/mouse) three times per week for an additional 20 weeks to induce TGF- ß1. All mice were at least 8 weeks of age and had developed at least one tumor when enrolled in the monitoring portion of this study (Figure 2). Forty mice were evaluated as part of this study, twenty transgenic animals and twenty nontransgenic controls. Methods: For the intra-abdominal tumor study, 7 week old female C57BL/J mice were inoculated intraperitoneally with 5 x 10 3 C6VL tumor cells (Figure 4). They were next treated with monoclonal antibodies to the C6VL tumors to assess survivability, as per the IACUC approved protocol of our collaborator. For the purposes of this study, mice were maintained until they showed clinical signs of morbidity (e.g. anorexia, hunched posture, non-responsive to stimuli), then they were humanely euthanized by carbon dioxide asphyxiation for tissue collection. All of the intra-abdominal model mice were scored three times per week. As tumors were not visible, no additional parameters regarding tumor size or character were recorded. Forty mice were evaluated as part of this study, twenty treated animals and twenty nontreated controls. This project was supported by funds from Johns Hopkins Center for Alternatives to Animal Testing, the Portland VA Research Foundation, and Merck Merial. Special thanks to Bryan Bustamante, Kurt Gritman, Kim Villines, Carla Webb, and Rachel Luksic for their assistance with data collection. Introduction Inter-Observer Variability Skin Tumors Acknowledgements Abdominal Tumors Use of Body Condition Scoring as an Adjunct Endpoint for Tumor Growth Studies D. Hickman,* VA Medical Center, Portland, OR Use of Body Condition Scoring as an Adjunct Endpoint for Tumor Growth Studies D. Hickman,* VA Medical Center, Portland, OR Figure 2: Representative example of mouse with skin papillomas, utilized for the skin tumor phase of this study. Figure 3: Average weight and body condition score for skin tumor study mice over 9-week observation period. Results & Discussion: We found that the use of body condition scoring as an adjunct means of monitoring well-being of animals where skin tumors have been induced was unrewarding. Despite the presence of up to 20 papilloma tumors on individual mice, the body condition score did not decrease in advance of other parameters, such as tumor size or ulceration (Figure 3). Clinically, ulcerated tumors became larger and developed secondary infections requiring euthanasia of the mouse prior to declines in body condition scores. In our experience, mice with ulcerated tumors showed consistently clinical signs historically used as endpoints for these types of studies (e.g. hunched posture, decreased appetite, reluctance to ambulate) and the decision to euthanize could be made when these signs were observed. Body condition scoring did not supply additional information of use. Figure 5: Average weight and body condition score for intra-abdominal tumor study mice over a 3-week (three times per week) observation period. Results & Discussion: Comparison of the initial and final weights and body condition scores showed that the weights increased while the body condition scores decreased (Figure 5). One-way analysis of variance was used to analyze the difference between control and treatment groups. A within group comparison was also made for weights and body condition scores of animals euthanized versus animals that died. This analysis showed that the means and confidence intervals for both groups were nearly identical suggesting that body condition score could reliably be used to intervene before morbidity progressed to mortality. Mice that developed intra-abdominal tumors showed an increase in weight of approximately 16% from initial when the initial and final weights of treated animals were compared to the control group. These mice also displayed a significant decline of at least one body condition score when the initial and final body condition scores of treated animals were compared to the control group. This affirms the hypothesis that fat and muscle loss occurs on these studies, even as animal weight is increasing. The data also showed that when a score dropped one level, the animal exhibited increased chances of morbidity. However, we were unable to conclude that body condition score alone is useful used as an endpoint for these studies. We collected subjective data regarding animal posture and attitude, but were unable to analyze this data in comparison to the body condition score. We noted that animals that developed a score of 1.5 began to show hunched posture and lack of coordination prior to death, but the intra-observer interpretation of these criteria were too varied. Future studies will evaluate objective scales of assessment for these assessments and compare with the body condition score. We anticipate that further study will show that the body condition score proves a sensitive means of alerting the investigator and veterinary staff that death is approaching, allowing them to focus on the evaluation of other signs, such as hydration, posture, and appearance, when making the determination of appropriate time for euthanasia. Figure 1: BCS chart from Ullman-Cullere et al (1999). Figure 4: Representative example of mice with distended abdomens, utilized for the intra- abdominal tumor phase of this study.